BACKGROUND: Contaminated surfaces play a crucial role in transmitting infectious diseases. Effective surface disinfection is essential for interrupting disease transmission by eliminating pathogens on these surfaces. While traditional chemical disinfectants are effective, they pose significant health and environmental concerns, prompting the search for natural alternatives. OBJECTIVE: To evaluate the efficacy of vinegar and asafoetida solution in surface disinfection. METHODS: The swab test was conducted on 10 different surfaces, including rough and smooth surfaces, contaminated with organisms such as Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus cereus, Candida albicans, and Aspergillus niger. Samples were collected before and after applying the test solution. Additionally, a real-settings swab test was performed on 20 sites with diverse textures within hospital and workplace settings to assess the practical applicability of the test solution in reducing microbial contamination. SETTING: The study was conducted in laboratory settings, followed by real-world application in hospital and workplace environments. INTERVENTION: The test solution, composed of vinegar and asafoetida, was applied to contaminated surfaces to assess its disinfection efficacy. Positive controls included 1% sodium hypochlorite and 5% phenol, while distilled water served as the negative control. PRIMARY OUTCOME MEASURE: The primary outcome was the reduction in microbial colony counts after the application of the test solution, quantified through swab samples before and after disinfection. RESULTS: Significant reductions in microbial colonies were observed across all tested surfaces following the application of the test solutions, with complete elimination achieved on many surfaces. Percent reductions in microbial colonies ranged from 50% to 100%, and log reductions varied from 0.2 to undefined i.e., complete reduction. CONCLUSION: The test solution effectively reduced microbial loads on smooth and rough surfaces, demonstrating its potential as a natural disinfectant. However, its long-term residual activity and efficacy against viral strains were not evaluated. Therefore, future research should address these gaps to optimize the application of the solution.